BACKGROUND: DNA repair is essential to protect from genomic instability and malignant transformation. Yet, tumor growth itself relies on efficient DNA repair to mitigate the detrimental impact of DNA damage associated with excessive cell division. Consistent with this, defects in key tumor suppressors such as the HR factor BRCA2, are not only linked to genome instability and tumorigenesis, but are also associated with an increase in tumor cell susceptibility to genotoxic stress. Modulating BRCA2 function may, thus, provide an important avenue to manipulate malignant growth, particularly in BRCA2-proficient tumors. RESULTS: We have identified the ubiquitin-specific protease USP21 as a positive regulator of BRCA2 stability. USP21 interacts with, deubiquitinates and stabilizes BRCA2 to promote efficient Rad51 accumulation at DSBs. As a result, depletion of USP21 decreases HR efficiency, causes an increase in DNA damage load and impairs tumor cell survival. Importantly, BRCA2 overexpression partially restores the USP21-associated survival defect. Biochemical dissection reveals that USP21 associates with the C-terminal OB domain of BRCA2, and consistent with this, a tumor cell line with a C-terminal BRCA2 truncation is unresponsive to USP21 depletion with regard to both BRCA2 stability and tumor growth. While BRCA2 was previously found to be ubiquitinated, USP21 is, to our knowledge, the first DUB directly involved in BRCA2 stabilization. However, although our data point to BRCA2 as the primary target of USP21 stabilization, we can at this point not rule out a direct effect of USP21 on other HR mediators. Importantly, we provide functional evidence for USP21 as a modulator of DSB repair by HR but not NHEJ. The repair pathway-specific impact of USP21 loss stands in contrast to the rather broad repair defects observed following depletion of many other DUBs, which affect DDR events associated with both NHEJ and HR. USP21 may, thus, present a unique target for the manipulation of HR, particularly in the context of tumor growth. The latter is of clinical relevance as there are few specific HR inhibitors known to date. Underlining the physiological relevance of our findings, USP21 is significantly overexpressed in hepatocellular carcinoma (HCC) patients and inversely correlates with patient survival. Moreover, elevated USP21 levels are linked to increased somatic copy number alterations, which are often a result of aberrant, non-allelic HR. Mechanistically, we show that loss of USP21 in HCC cell lines results in a reduction of BRCA2, increased DNA damage accumulation and a concomitant, BRCA2-dependent growth defect. The latter is consistent with a protective role for USP21, likely involving HR to counteract excessive replication-associated DNA damage, cell cycle arrest and/or apoptosis, which may, at least in part, account for the observed increase in HCC aggressiveness (poor survival). IMPLICATIONS: Together, our findings identify deubiquitination as a novel means to regulate BRCA2 function and point to USP21 as a potential therapeutic target, particularly in light of recent efforts to develop specific small molecule DUB inhibitors/activators. In light of the central role of BRCA2 during replication stress, it is tempting to speculate that USP21 loss may affect additional BRCA2-proficient tumor types. Consistent with this, we found that 27 % of tumors available for TCGA analysis showed a significant increase in USP21 expression. This work has been accepted for publication in Nature Communications. Given that our findings point to a role in DNA repair that is largely unrelated to alterations in chromatin structure, USP21 may have limited implications for DNA damage-associated epigenetic dysfunction and will not be a focus of our future research program.